Abstract

Transposable element activity is repressed in the germline in animals by PIWI-interacting RNAs (piRNAs), a class of small RNAs produced by genomic loci mostly composed of TE sequences. The mechanism of induction of piRNA production by these loci is still enigmatic. We have shown that, in Drosophila melanogaster, a cluster of tandemly repeated P-lacZ-white transgenes can be activated for piRNA production by maternal inheritance of a cytoplasm containing homologous piRNAs. This activated state is stably transmitted over generations and allows trans-silencing of a homologous transgenic target in the female germline. Such an epigenetic conversion displays the functional characteristics of a paramutation, i.e., a heritable epigenetic modification of one allele by the other. We report here that piRNA production and trans-silencing capacities of the paramutated cluster depend on the function of the rhino, cutoff, and zucchini genes involved in primary piRNA biogenesis in the germline, as well as on that of the aubergine gene implicated in the ping-pong piRNA amplification step. The 21-nt RNAs, which are produced by the paramutated cluster, in addition to 23- to 28-nt piRNAs are not necessary for paramutation to occur. Production of these 21-nt RNAs requires Dicer-2 but also all the piRNA genes tested. Moreover, cytoplasmic transmission of piRNAs homologous to only a subregion of the transgenic locus can generate a strong paramutated locus that produces piRNAs along the whole length of the transgenes. Finally, we observed that maternally inherited transgenic small RNAs can also impact transgene expression in the soma. In conclusion, paramutation involves both nuclear (Rhino, Cutoff) and cytoplasmic (Aubergine, Zucchini) actors of the piRNA pathway. In addition, since it is observed between nonfully homologous loci located on different chromosomes, paramutation may play a crucial role in epigenome shaping in Drosophila natural populations.

GENOMES must confront the presence of a large fraction of mobile DNA whose activity can result in severe deleterious effects on chromosome stability and gametogenesis. In the germline of animals, a system of genomic traps exists into which any transposable element (TE) can insert, thereby generating loci that contain a catalog of potentially dangerous sequences that have to be repressed (Brennecke et al. 2007; Pane et al. 2011; Iwasaki et al. 2015). In the Drosophila melanogaster germline, most of these loci are transcribed in both directions (dual-strand clusters) and undergo noncanonical transcription and RNA processing (Mohn et al. 2014; Zhang et al. 2014). This results in production of noncoding small RNAs having the capacity to target the transcripts of the homologous, potentially active, TE copies scattered throughout the genome. These small RNAs are called PIWI-interacting RNAs (piRNAs) and repress TE activity at both the transcriptional and post-transcriptional levels (Sato and Siomi 2013; Weick and Miska 2014). piRNA biogenesis in the germline involves a nuclear and a cytoplasmic step. In the nucleus, piRNA-producing cluster transcription requires in most of the cases the presence of the HP1 paralog Rhino associated with Deadlock and Cutoff on the locus, forming the so-called RDC complex (Klattenhoff et al. 2009; Pane et al. 2011; Mohn et al. 2014; Zhang et al. 2014). In the cytoplasm, transcripts produced by the piRNA locus are sliced in an optically dense region surrounding the nucleus, called the nuage, and small RNAs (23–28 nt) loaded on Piwi or Aubergine proteins are produced (primary piRNAs). Further, piRNAs loaded on Piwi enter the nucleus and target euchromatic TE copies to induce their transcriptional repression via heterochromatin formation, which involves HP1 (Wang and Elgin 2011; Sienski et al. 2012; Le Thomas et al. 2013; Rozhkov et al. 2013). In contrast, piRNAs loaded on Aubergine remain in the nuage and target homologous transcripts being exported from the nucleus, which are produced by both homologous TE copies and the piRNA locus. This produces secondary piRNAs loaded on Aubergine or Ago3, other PIWI proteins (Weick and Miska 2014; Iwasaki et al. 2015) and results in a piRNA amplification process called ping-pong amplification (Brennecke et al. 2007; Gunawardane et al. 2007). Finally, piRNA loaded on Piwi can also be produced downstream of the ping-pong amplification step by a slicing process that can spread on targeted RNA, increasing both piRNA quantity and diversity (Han et al. 2015; Mohn et al. 2015; Siomi and Siomi 2015). Upstream of this complex machinery, the presence of Rhino on the piRNA-producing locus is particularly important since it appears sufficient to promote processing of transcripts by the piRNA machinery (Klattenhoff et al. 2009; Zhang et al. 2014). How Rhino is addressed to a piRNA-producing locus is still unclear.

In Drosophila, in contrast to Caenorhabditis elegans (Ruby et al. 2006; Batista et al. 2008), the production of piRNAs by a piRNA locus in the germline does not appear to be only genetically determined, i.e., no specific sequence motif or structure has been identified that is sufficient to promote piRNA production by DNA adjacent to this sequence. Conversely, using piRNA-producing loci that repress P-transposable elements (Ronsseray et al. 1996), it was shown that maternal transmission of piRNAs together with the piRNA locus can stimulate production of piRNAs by paternally inherited P-elements scattered through the genome (Brennecke et al. 2008). In addition, analysis of ageing effects on I-transposable element repression capacities showed that the amount of I-homologous piRNAs in adult ovaries is correlated to the amount of homologous piRNAs deposited in embryos (Grentzinger et al. 2012). Furthermore, it was shown that de novo, long-term activation of a piRNA locus can be achieved in Drosophila by maternal transmission of homologous piRNAs, without transmission of the initial piRNA donor locus. Under these conditions, there is emergence of a new autonomous and stable piRNA locus (de Vanssay et al. 2012). Indeed, a transgenic cluster of P-element deriving transgenes (called BX2), inert for repression and piRNA production, can be activated by crossing with females bearing a transgenic cluster (called T-1) that produces abundant piRNAs in both orientations homologous to the BX2 cluster (de Vanssay et al. 2012). Once activated, this new piRNA cluster, called BX2*, is stable over generations (n > 100) in absence of the inducer T-1 locus and is able to produce abundant transgenic sense and antisense piRNAs. Cytoplasm of oocytes produced by these females can again de novo activate piRNA production by a paternally inherited inactive BX2 cluster (de Vanssay et al. 2012). This recurrent epigenetic conversion process presents all the features of a “paramutation” process, a phenomenon previously described in plants (Brink 1956; Coe 1959) and recently in worms (Shirayama et al. 2012; Sapetschnig et al. 2015), and described as “an epigenetic interaction between two alleles of a locus, through which one allele induces a heritable modification of the other allele without modifying the DNA sequence” (Brink 1956; Chandler 2007). It was shown further that a BX2* locus has a modified chromatin structure, showing an increase in H3K9 methylation with regard to the nonparamutated BX2 locus (Le Thomas et al. 2014). This example of paramutation thus results in communication within a genome between homologous clusters allowing one cluster to transfer piRNA production capacity to a previously inert cluster. Paramutation may play an important role in establishment of TE repression following a genetic invasion since master sites of repression could activate piRNA secondary sites thus reinforcing their repressive capacity. Investigating the mechanism of de novo activation of piRNA-producing loci is therefore important for understanding aspects of epigenome shaping in general.

We have therefore analyzed the functional requirements and properties of a BX2* paramutated locus. This locus allows the investigation of the autonomous properties of a piRNA-producing locus, i.e., independently of the influence of homologous sequences present in the genome that could interfere with the process. The BX2* locus was previously shown to produce, not only abundant transgenic piRNAs, but also 21-nt RNAs inferred to be small-interfering RNAs (siRNAs), but the precise nature of these 21-nt RNAs and their functional role in silencing capacities were not elucidated. Here, we show first that BX2* silencing and piRNA production capacities depend on the rhino and cutoff genes, involved in the noncanonical transcription of piRNA-producing loci, and on the aubergine and zucchini genes, involved in piRNA cytoplasmic processing. In addition, transgenic 21-nt RNA production depends on Dcr-2, which characterizes them as siRNAs but they are not necessary for paramutation to occur. Surprisingly, production of these siRNAs depends also on the piRNA pathway. Moreover, paramutation can occur between only partially homologous loci within a genome and is accompanied by a rapid spreading of the capacity to produce piRNAs to the nonhomologous sequences within the paramutated locus. Finally, an impact of maternally inherited small RNAs linked to paramutation is also observed in somatic cells. These findings allow us to characterize more precisely the molecular mechanism of paramutation in animals and to propose that it is a key factor in epigenome shaping in natural populations.

Materials and Methods

Experimental conditions

All crosses were performed at 25° and involved three to five couples in most cases. lacZ expression assays were carried out using X-gal overnight staining as described in Lemaitre et al. (1993), except that ovaries were fixed for 8 min. Trans-silencing was quantified as previously described by determining the percentage of egg chambers with no lacZ expression in the germline (Josse et al. 2007). It is referred to as the trans-silencing effect percentage (TSE %).

Pigment dosage

Head pigments were quantified as described in Weiler and Wakimoto (2002). For each genotype, three replicates were measured. For each replicate, five heads of 4- to 6-day-old females were manually dissected for the dosage. Absorbance was measured at 480 nm.

Transgenes and strains

P-lacZ fusion enhancer-trap transgenes: P-1152 and BQ16, contain an in-frame translational fusion of the Escherichia coli lacZ gene to the second exon of the P-transposase gene and a rosy transformation marker (O’Kane and Gehring 1987). The P-1152 insertion was mapped to the telomere of the X chromosome (cytological site 1A) and consists of two P-lacZ insertions in the same telomeric-associated sequence unit (TAS) (Karpen and Spradling 1992) and in the same orientation (Josse et al. 2007). P-1152 is homozygous viable and fertile. BQ16 is located at 64C in euchromatin of the third chromosome and is homozygous viable and fertile. P-1152 shows no lacZ expression in the ovary; BQ16 is strongly expressed in the nurse cells and in the oocyte (Josse et al. 2007). RS3 is a P-FRT-white transgene (FBtp0001534). It is inserted in the TAS of the 3R chromosomal arm (cytological site 100E3). It is homozygous viable and fertile (DGRC, Kyoto no. 123282). P-1152 and RS3 induce a strong TSE and produce abundant ovarian transgenic piRNAs (Roche and Rio 1998; Josse et al. 2007; de Vanssay et al. 2012; Dufourt et al. 2014) and this study (see Figure 3A). The Lk-P(1A) line carries two full-length P-elements inserted in TAS at the X-chromosome telomere (Ronsseray et al. 1991). These telomeric P-insertions derive from a chromosome present in a natural population (Biemont et al. 1990) and induce very strong repression of P-element transposition and P-induced hybrid dysgenesis (Ronsseray et al. 1991, 1996). Lk-P(1A) females produce abundant P-homologous ovarian piRNAs (Brennecke et al. 2008). The repressive capacity of the Lk-P(1A) line was verified immediately prior to the experiments reported in Supporting Information, Figure S6 and a very strong repression capacity was found (data not shown).

P{lacW} clusters:

The BX2 line carries seven P-lacZ-white transgenes, including at least one defective copy, inserted in tandem and in the same orientation at cytological site 50C on the second chromosome (Dorer and Henikoff 1994). The transgene insertion site is located near the mRpL53 gene in an Ago1 intron (de Vanssay et al. 2012). This site is not a piRNA-producing locus, as observed for instance in the deep-sequencing dataset from P-1152 or w1118 ovaries (data not shown). The P-lacZ-white construct contains the P-lacZ translational fusion and is marked by the mini-white gene (P{lacW}, FBtp0000204, 10,691 bp). The T-1 line derives from X-ray treatment of BX2 (Dorer and Henikoff 1997). T-1 has chromosomal rearrangements including translocations between the second and third chromosomes. DX1 was generated during the same P-lacZ-white transposase-mediated remobilization at 50C than BX2 and carries six P-lacZ-white transgenes, one of which is in opposite orientation to the others, at the same genomic site as BX2 (Dorer and Henikoff 1994, 1997). After overnight staining, weak lacZ expression is detected in the follicle cells of BX2, T-1, and DX1 female ovaries, presumably because of a position effect at 50C, but no staining is observed in the germline (data not shown). The three P{lacW} clusters show variegated repression of white in the eyes due to repeat induced gene silencing (RIGS) (Dorer and Henikoff 1994, 1997). The extent of variegated repression of white is different between the clusters (T-1 >> DX1 > BX2). The BX2 cluster was not used to assay the somatic phenotypic impact of piRNA maternal inheritance since RIGS-induced repression is too low in this case.

Lines carrying transgenes have M genetic backgrounds (devoid of P-transposable elements), as do the multimarked balancer stocks used in genetic experiments. The w1118 and Cantony lines were used as controls completely devoid of any P-element or transgene. Crosses performed with BX2, T-1, or DX1 were performed with females carrying the cluster at the heterozygous state because of the sterility (BX2, DX1) and lethality (T-1) induced by transgene clusters.

Two strong hypomorphic mutant alleles of aubergine (aub) induced by EMS were used. Both of them are homozygous female sterile and TSE was previously shown to be abolished by a heteroallelic combination of these alleles (Josse et al. 2007). aubQC42 comes from the Bloomington Stock Center (stock no. 4968) and has not been characterized at the molecular level (Schupbach and Wieschaus 1991). aubN11 has a 154-bp deletion, resulting in a frameshift that is predicted to add 16 novel amino acids after residue 740. Dicer-2L811fsX (Dcr-2L811fsx) is a loss-of-function allele induced by EMS, which has a sequence variant at residue 811 resulting in a stop codon (Lee et al. 2004). It is homozygous viable and fertile. rhino02086 (rhi2) results from a P{PZ} insertion (P-lacZ-rosy) at nucleotide 267. The P{PZ} transgene is oriented in the opposite direction to that of rhi transcription (Volpe et al. 2001) (Bloomington no. 12226) . rhiKG00910 (rhiKG) is due to the insertion of a P{SUPor-P} transgene, which carries the yellow and white transformation markers (Gene Disruption Project 2001, Bloomington no. 13161). cutoffWM25 (cuffWM25) and cuffQQ37 alleles were isolated from an EMS screen (Schupbach and Wieschaus 1991). cuffWM25 corresponds to the replacement of the first methionine by a lysine and cuffQQ37 has a T to A substitution at position 786 that could result in a splicing alteration (data not shown). zucchini (zuc) alleles were isolated from an EMS screen (Schupbach and Wieschaus 1991). zucHM27 contains a stop codon at residue 5 and zucSG63 a substitution of histidine 169 with a tyrosine in the conserved HKD domain presumably involved in nuclease activity. TSE was previously shown to be abolished by a heteroallelic combination of these alleles (Todeschini et al. 2010). The crosses performed to generate heteroallelic mutant BX2* females are indicated in Table S1. When information was available, the most severe allele was introduced maternally. Lines carrying mutations of zuc and cuff were kindly provided by Attilio Pane and Trudi Schüpbach and aubN11 was kindly provided by Paul Macdonald.

All the alleles described above are located on the second chromosome and are maintained over a Cy balancer chromosome. Additional information about mutants and stocks are available at FlyBase: http://flybase.bio.indiana.edu/.

Deep sequencing analyses

A small RNA fraction, from 18 nt to 30 nt in length, was obtained by separating it on a denaturing polyacrylamide gel from total RNA extracted from dissected ovaries. This fraction was used to generate multiplexed libraries with Illumina TruSeq Small RNA Library preparation kits (RS-200-0012, RS200-0024, RS-200-036, or RS-200-048) at Fasteris (http://www.fasteris.com). A house protocol based on TruSeq, which reduces 2S RNA (30 nt) contamination in the final library, was performed (except for libraries whose GRH number is below no. 40). Libraries were sequenced using Illumina HiSequation 2000 and 2500. Sequence reads in fastq format were trimmed from the adapter sequence 5′-CTGTAGGCACCATCAATCGTA-3′ (GRH12, GRH13, GRH14, GRH17) or 5′-TGGAATTCTCGGGTGCCAAG-3′ (other samples) and matched to the D. melanogaster genome release 5.49 using Bowtie (Langmead et al. 2009) and to the sequences of the P-element constructs P{lacW} (FlyBase ID FBtp0000204) as indexed reference. Only 19- to 29-nt reads matching the reference sequences with 0 or 1 mismatch were retained for subsequent analysis. For global annotation of the libraries (Table S1), we used the release 5.49 of fasta reference files available in FlyBase, including transposon sequences (dmel-all-transposon_r5.49.fasta) and the release 20 of miRNA sequences from miRBase (www.mirbase.org).

Sequence length distributions, small RNA mapping, and small RNA overlap signatures were generated from bowtie alignments using Python and R (www.r-project.org/) scripts, which were wrapped and run in a Galaxy instance publicly available at http://mississippi.fr. Tools and workflows used in this study may be downloaded from this Galaxy instance. For library comparisons, read counts were normalized (Table S1) to the total number of small RNAs that matched the D. melanogaster genome (release 5.49) and did not correspond to abundant cellular RNAs (tRNAs and miscRNAs). For small RNA mapping (Figure 1, Figure 2, and Figure 3 and Figure S1, Figure S3, and Figure S6), we took into account only RNA reads that uniquely aligned to P{lacW} or the 42AB locus.

Effect of mutations affecting the piRNA or siRNA pathways on trans-silencing and small RNA production capacities of a BX2* cluster. Upper box: Mating scheme used to analyze the effect of mutations (mut) affecting aubergine, Dicer-2, rhino, cutoff, and zucchini on BX2* ovarian small RNA production and trans-silencing capacities. As the BX2 cluster, all genes are located on chromosome 2. (Left) Heterozygous BX2* aub−, BX2* Dcr-2−, BX2* rhi−, BX2* cuff−, or BX2* zuc− females were crossed with heterozygous males carrying a mutant allele of the same gene to generate either heterozygous control or loss-of-function females for deep sequencing of ovarian small RNAs. (Right) The same heterozygous females were crossed with heterozygous males carrying a mutant allele of the same gene plus a P-lacZ target transgene (BQ16) to measure the effect of the mutation tested on BX2* trans-silencing capacities (TSE %). TSE was quantified by determining the percentage of egg chambers with no lacZ expression in the germline. In the genotypes presented, maternally inherited chromosomes (chromosomes 2 and 3) are indicated above the bar. Cy is a balancer chromosome carrying a dominant phenotypic marker. (A–L) In each case, the genotype tested is indicated, and the percentage of TSE is given below the genotype with the total number of egg chambers assayed in parentheses. Nonparamutated BX2 (“naive”) and BX2* at generation 83 were analyzed as controls. Histograms show the length distributions of ovarian small RNAs matching the P{lacW} locus. Positive and negative values correspond to sense and antisense reads, respectively. Plots show the abundance of 19- to 29-nt small RNAs matching the P{lacW} locus. Analysis of the effect of aub, rhi, cuff, and zuc mutations shows that BX2* silencing capacities, ovarian P{lacW} 23- to 28-nt piRNA, but also 21-nt RNA production depend on the piRNA pathway. Dcr-2 loss of function strongly affects production of P{lacW} homologous 21-nt RNAs without affecting that of 23- to 28-nt RNAs or trans-silencing capacities. P{lacW} 21 nt are not necessary for the maintenance of the paramutated state and their biogenesis depends on both Dcr-2 and the piRNA pathway. TSE results for Dcr-2 mutants (E and F) are reprinted from de Vanssay et al. (2012).

Paramutation by a cytoplasm devoid of siRNAs. (A) BX2* Dcr-2−/− females were crossed to BX2 Dcr-2+/+ (naive) males to generate G1 females having paternally inherited a BX2 locus and maternally inherited piRNAs but no 21-nt siRNAs homologous to the P{lacW} cluster. In addition, these G1 females paternally inherited a Dcr-2+ allele. These G1 females were crossed with Dcr-2+ males to establish a BX2* Dcr-2+/Dcr-2+ line in which no maternal P{lacW} siRNAs have been initially introduced in G0. Cy is a balancer chromosome carrying a dominant phenotypic marker. Maternal chromosomal complement is written above the bar. (B) At generations 0, 1, and 2, deep sequencing of ovarian small RNAs was performed and silencing capacity was controlled in parallel by crossing females with males carrying the BQ16 target transgene and scoring TSE in female progeny. The genotype tested is indicated and TSE percentages and ovarian 19- to 29-nt RNAs are presented as in Figure 1. As expected, G1 and G2 females show complete silencing capacities. The same was true for successive Gn generations (G6, TSE = 100%, n = 450; G10, TSE = 100%, n = 1100). Despite the lack of maternal P{lacW} siRNA maternal transmission in G0, such ovarian small RNAs appear already in G1 females and increase in G2 in BX2* Dcr-2+/+ females (dashed arrow). Therefore, maternal inheritance of P{lacW} homologous piRNAs, but no siRNAs, results in a strong and stable paramutated BX2* locus and in immediate production of both P{lacW} piRNAs and siRNAs in females carrying a Dcr2+ allele.

Paramutation of a BX2 locus by partially homologous piRNAs loci. BX2 males carrying a P{lacW} naive cluster were crossed to females carrying partially homologous P-transgenes inserted in telomeric piRNA-producing loci at the heterozygous state. Female progeny were recovered that carry the BX2 locus but did not carry the telomeric transgenes. These G1 females inherited a cytoplasm carrying piRNA homologous to the telomeric transgenes, which therefore cover only partially the BX2 P{lacW} transgene sequence. Lines were established and the trans-silencing capacities of these putative BX2* lines were tested in subsequent generations, as in Figure 1 and Figure 2. In addition, deep sequencing of ovarian small RNAs was performed in G3, G5, and G10. (A, left and middle) Structure of the P-1152 and RS3 telomeric P-transgenes used and of the BX2 P{lacW} transgenes. Colored thick lines below the transgenes indicate the sequences shared by the P{lacW} and telomeric transgenes. The P-1152 line carries two P-lacZ-rosy (P{lArB}) transgenes inserted in the telomeric associated sequences (TAS) of the X chromosome. RS3 carries a P-FRT-white transgene in the TAS of the 3R chromosomal arm. These two lines induce a strong TSE. (A, right) Ovarian piRNA production in females that carry a maternally inherited P-1152 or RS3 locus at the heterozygous state (G1 females from crosses between P-1152 and RS3 females with males devoid of transgene) is mapped on the P{lacW} transgene. (B) Three types of replicate BX2* sublines were generated: Sublines that inherited in G1 (1) a cytoplasm from females heterozygous for the P-1152 locus (P-1152BX2 sublines, in orange); (2) a cytoplasm from females heterozygous for the RS3 locus (RS3BX2 sublines, in green); and (3) a cytoplasm from females heterozygous for both the P-1152 and RS3 loci (P-1152+RS3BX2 sublines, in purple). Trans-silencing capacities of various BX2* lines are shown with regard to generations (TSE %, n > 500 in all assays). Names of replicate sublines are given below the graphs. (C) Deep sequencing of ovarian small RNAs in various BX2* sublines at generations 3, 5, and 10. Plots show the abundance of 19- to 29-nt small RNAs matching P{lacW}. TSE assays show that silencing capacities vary among replicate BX2* lines but strong and stable paramutated lines can be recovered with the three types of G0 cytoplasmic inheritance (B). Ovarian small RNAs were analyzed for a subset of the BX2* lines showing strong silencing capacities. The name of the line is indicated on the left of the graph. Deep sequencing analysis shows that piRNAs corresponding to the whole length of P{lacW} can be produced as early as generation 3 (C), showing that piRNA production by a paramutated locus can rapidly extend to the part of the locus that did not originally receive maternally inherited piRNAs.

Distributions of piRNA overlaps (ping-pong signatures, Figure S2 and Figure S5) were computed as first described in Klattenhoff et al. (2009) and detailed in Antoniewski (2014). Thus, for each sequencing dataset, we collected all the 23- to 28-nt RNA reads matching P{lacW} or the 42AB locus whose 5′ ends overlapped with another 23- to 28-nt RNA read on the opposite strand. Then, for each possible overlap of 1–28 nt, the number of read pairs was counted. Distributions of siRNA overlaps (Figure S3) were computed using a similar procedure, except that 20- to 21-nt RNA reads were collected instead of the 23- to 28-nt RNA reads. The distributions of piRNA/siRNAs overlaps (Figure S3) were computed by collecting separately the 20- to 21-nt and 23- to 28-nt RNA reads matching P{lacW} or the 42AB locus and counting for each possible overlap of 1–21 nt the number of read pairs across these two distinct read datasets. To plot the overlap signatures, a z-score was calculated by computing, for each overlap of 1 to i nucleotides, the number O(i) of read pairs and converting it, using the formula z(i) = (O(i)-mean(O))/standard deviation (O).

Data availability

Small RNA sequences have been deposited at the European Nucleotide Archive (ENA) (http://www.ebi.ac.uk/ena) under accession number PRJEB11491.

Results

Effect of mutation affecting the piRNA or siRNA pathway on BX2* small RNA production and silencing capacities

Paramutated BX2* females were previously shown to produce 23- to 28-nt and 21-nt ovarian sense and antisense small RNAs homologous to the cluster of P-lacZ-white transgenes (called P{lacW}) and to induce complete silencing of homologous transgene expression in the female germline (de Vanssay et al. 2012). This homology-dependent silencing, which reflects piRNA functionality, is quantified using the trans-silencing effect (TSE) assay (Josse et al. 2007) in which the percentage of repressed egg chambers is measured. The 23- to 28-nt, but not 21-nt, RNAs presented the molecular signature of the ping-pong amplification step of the piRNA pathway. This strongly suggests that production of the 23- to 28-nt RNAs is dependent on the piRNA pathway, whereas that of the 21-nt RNAs would depend on the siRNA pathway. Moreover, the TSE capacities of the BX2* paramutated locus were shown to be completely impaired by loss of function of aubergine (aub) involved in the piRNA ping-pong step, whereas they were insensitive to loss of function of Dicer-2 (Dcr-2) involved in the siRNA pathway (de Vanssay et al. 2012). This suggested that the 21 nt would not be necessary for BX2* silencing capacities.

We thus performed deep sequencing of ovarian small RNAs of mutant females for aub and Dcr-2 and extended the analysis to rhino (rhi) and cutoff (cuff), which are nuclear actors involved in piRNA loci noncanonical transcription (Mohn et al. 2014; Zhang et al. 2014), and to zucchini (zuc), which is involved in the cleavage of piRNA precursors and secondary piRNAs in the cytoplasm (Ipsaro et al. 2012; Nishimasu et al. 2012; Voigt et al. 2012; Han et al. 2015; Mohn et al. 2015). All these genes are located, as BX2 is, on chromosome 2. For each gene tested, a recombinant chromosome carrying a BX2* locus and a mutated allele was generated. Females heterozygous for this recombinant chromosome were crossed with heterozygous males, carrying one mutant allele of the corresponding tested gene, to generate control heterozygote, loss-of-function heteroallelic (aub, rhi, cuff, zuc) and homozygote (Dcr-2) females for deep sequencing of ovarian small RNAs (Figure 1, upper box, and Table S1). In addition, females heterozygous for recombinant chromosomes were crossed with heterozygous males carrying a corresponding mutated allele and a target P-lacZ transgene expressed in the female germline to investigate the effect of the mutation tested on the trans-silencing capacities of BX2* (Figure 1, upper box). As controls, we analyzed small RNAs from ovaries of BX2* females at generation 83 (G83) after the paramutation process and of BX2 nonparamutated females (called “BX2 naive”). BX2* lines maintained at 25° show a very strong stability through generations, as tested every five generations, showing a complete repression capacities (for example G83: TSE = 100%, n = 1500; G115: TSE = 100%, n = 1150). Figure 1 shows control females together with heterozygous and homozygous females for loss-of-function mutations for each gene tested, in terms of their capacity for trans-silencing (TSE %) and for production of ovarian small RNAs homologous to the P{lacW} transgene. As expected, the BX2 naive cluster showed no silencing capacities (TSE = 0%, n = 2000) and did not produce significant amounts of P{lacW} homologous 23- to 28-nt or 21-nt RNAs (Figure 1A) whereas the BX2* paramutated cluster continued to induce complete trans-silencing and to produce high levels of P{lacW} homologous 23- to 28-nt and 21-nt RNAs 83 generations after the paramutation process (Figure 1B). aub loss of function resulted in the complete loss of 23- to 28-nt RNAs homologous to the P{lacW} transgene (Figure 1D), when compared to heterozygous aub females (Figure 1C). Surprisingly, aub loss of function also affected production of the 21-nt RNAs by the P{lacW} cluster (Figure 1D). This did not result from a genomic background effect on the siRNA pathway in aub mutant females since the production of endogenous siRNAs at the esi-1 locus was not affected (Figure S4, D). Dcr-2 loss of function resulted in a strong decrease of 21-nt RNAs homologous to the P{lacW} transgene but the production of corresponding 23- to 28-nt RNAs was not affected, nor their ping-pong signature (Figure 1F and Figure S2J). Analysis of mutations affecting rhi, cuff or zuc produced similar results to that of aub mutations. TSE percentages showed that rhi, cuff, or zuc loss of function resulted in loss of BX2* silencing capacities (Figure 1, H, J, and L), whereas no effect was found with rhi, cuff, and zuc heterozygotes (Figure 1, G, I, and K). rhi, cuff, and zuc loss of function also resulted in complete loss of ovarian P{lacW} piRNA production together with an almost complete loss of 21-nt RNAs homologous to the transgene (Figure 1, H, J, and L). As expected, these mutants exhibited no effect on the production of endogenous siRNAs at the esi-1 locus (Figure S4) but production of piRNAs by the 42AB typical dual-strand piRNA locus was affected (Figure S1). In conclusion, BX2* silencing capacities and P{lacW} homologous piRNA production depend on all the piRNA pathway genes tested, including nuclear actors, and do not depend on Dcr-2. Interestingly, Dcr-2-dependent 21-nt RNAs’ production by the cluster also depends on all the piRNA pathway genes tested. BX2* is thus a typical dual-strand piRNA cluster, requiring the primary and secondary piRNA pathways, which allows the study of the properties of a piRNA cluster, independently from any influence of endogenous homologous sequences in trans.

Production of ovarian transgenic siRNAs does not require siRNA maternal inheritance

Our previous work (de Vanssay et al. 2012), combined with the results in Figure 1F, shows that BX2* silencing properties and production of piRNAs homologous to the P{lacW} transgene do not require maternal inheritance of siRNAs. Results in Figure 1 show that presence of ovarian P{lacW} 21-nt siRNAs depends, in addition to Dcr-2, on the function of all piRNA pathway genes tested. We asked next if production of P{lacW} ovarian siRNAs requires maternal inheritance of such siRNAs. We thus generated BX2* females carrying a paternally inherited Dicer-2 wild-type allele and maternally inherited P{lacW} homologous piRNAs but no siRNAs, because their G0 mothers were homozygous for Dcr-2 mutations (Figure 2). As expected (de Vanssay et al. 2012), these G1Dcr-2 heterozygous females showed complete silencing capacities. Deep sequencing shows that, in addition to ovarian P{lacW} piRNAs, they produce P{lacW} siRNAs. Further, these G1 females were crossed with Dcr-2+/+ males and increased ovarian P{lacW} siRNA levels were found in BX2* G2 females having recovered a Dcr-2+/+ genotype. Therefore, production of ovarian P{lacW} siRNAs by females having a functional siRNA pathway does not require homologous siRNA inheritance and can be induced by inheritance of solely homologous piRNAs.

Paramutation of a locus only partially covered by maternally inherited piRNAs can be stable and is accompanied by cis-spreading of piRNA production capacity

Targeting of transgenes or natural transposable elements by piRNAs can lead to spreading of piRNA production to sequences adjacent to the targeted elements (Muerdter et al. 2011; Olovnikov et al. 2013; Shpiz et al. 2014). We tested if spreading could be observed for a silencer locus induced by paramutation. Telomeric transgenes inserted in subtelomeric heterochromatin can show strong trans-silencing capacities (Roche and Rio 1998; Ronsseray et al. 2003; Josse et al. 2007; Josse et al. 2008; Dufourt et al. 2014) and produce abundant piRNAs in the germline (Muerdter et al. 2011; de Vanssay et al. 2012). These transgenes have a different structure than the P{lacW} transgene of BX2 but share some common sequences (de Vanssay et al. 2012). It was therefore possible to produce females, having paternally inherited a BX2 naive locus and maternally inherited cytoplasm containing piRNAs homologous to only a subset of the P{lacW} transgene structure. Two telomeric transgenic loci were used (Figure 3). The P-1152 line carries two copies of the P-lacZ-rosy transgene (P{lArB}) in the telomeric associated sequences (TAS) of the X chromosome (Josse et al. 2007). The P-1152 telomeric locus was the canonical silencer locus used in previous trans-silencing effect studies (Roche and Rio 1998; Josse et al. 2007, 2008; Todeschini et al. 2010; Pöyhönen et al. 2012). RS3 carries a P-FRT-white transgene in the TAS of the third chromosome (3R arm). It also induces a strong TSE (Dufourt et al. 2014). Thus, BX2 P{lacW} transgenes shares P-element and lacZ sequences with P-1152 and shares P-element and white sequences with RS3. In both cases, a part of the P{lacW} sequence is missing in the telomeric transgenic construct (see Figure 3A). In order to transmit the cytoplasm, but not the telomeric transgenes, females heterozygous for these telomeric transgenes were generated. We further crossed BX2 males, from a nonparamutated line, with females heterozygous for each or both telomeric transgenes. G1 females having inherited the BX2 locus, but not the telomeric transgenes, were recovered and potentially paramutated BX2 lines were established and analyzed for trans-silencing and ovarian piRNA production capacities over generations. Figure 3B shows that G1 females that carried a paternally inherited BX2 cluster and recovered cytoplasm containing piRNAs produced by partially homologous transgenes, can show strong or complete silencing capacities. However, the efficiency was not as strong as that of paramutation experiments in which the P{lacW} transgenes of the BX2 paramutated locus inherit piRNAs homologous to the entire transgene length, which always result in a complete and stable paramutation (de Vanssay et al. 2012 and data not shown). For the BX2* lines activated by piRNAs homologous to the P-1152 transgenes (called P-1152BX2*), two lines (QA5 and QA6) showed very strong silencing capacities after >30–50 generations (QA5 is at 100% at each generation tested), which appeared even more stable that those reported in de Vanssay et al. (2012). One line (BB5) exhibited a period of strong silencing capacity followed by a reduction in silencing and one line showed partial silencing capacities (BB6). For the BX2* lines activated by piRNAs homologous to the P-white transgene (called RS3BX2*), one line (QA8) established a long-term strong level of repression, while the other line (BB7-4) showed diminishing repression over time. For the BX2* lines activated by piRNAs homologous to both P-1152 and RS3 transgenes (called P-1152 + RS3BX2*), the situation resembled that of lines activated by P-1152 cytoplasm, with one line showing complete repression capacity (QA1) and other lines showing intermediate repression levels. An additive effect was not observed since these P-1152 + RS3BX2* lines did not appear stronger than the P-1152BX2* lines despite the fact that small RNAs covering most of the sequence were maternally transmitted in G0. We conclude that paramutation by partially homologous transgenes is globally less efficient than paramutation by fully homologous transgenes. However, piRNAs produced by partially homologous transgenes can induce stable and strong paramutation we call “partially homologous paramutations.”

For each type of G0 maternal inheritance, lines showing strong silencing capacities were chosen and ovarian small RNA production was analyzed in G3, G5 and G10 by deep sequencing (Figure 3C and Figure S5). Figure 3C shows that, as early as generation 3, the three types of G0 maternal inheritance resulted in strong production of small RNAs covering the entire BX2 P{lacW} transgene length. Thus, partially homologous paramutations are associated with rapid and stable spreading of piRNA production by nonhomologous sequences of the targets, similarly to what was found for target transgenes (Muerdter et al. 2011; Olovnikov et al. 2013; Han et al. 2015; Mohn et al. 2015) or transposable elements (Shpiz et al. 2014).

Further, the paramutagenicity of P-1152BX2*, RS3BX2*, and P-1152+RS3BX2* females was tested. Figure 4 shows that maternal transmission of cytoplasm produced by these females, combined to paternal transmission of a BX2 naive locus, resulted in female progeny showing strong trans-silencing properties. These capacities are transmitted over generations, as tested until G36. Thus, paramutation mediated via maternal inheritance of piRNAs homologous to a subregion of the targeted locus can be strong and stable and can generate strongly paramutagenic loci.

Paramutagenicity of a partially homologous paramutation. BX2* lines having, at G0, maternally inherited piRNA homologous to a part of the BX2 P{lacW} transgene sequence, and showing strong silencing capacities at the 20th generation (Figure 3), were tested for their ability to de novo paramutate a naive BX2 locus. (A) The name of the line is indicated beside the G0 female genotype. Maternal chromosomal complement is written above the bar. Cy and CyRoi are balancer chromosomes devoid of transgenes and carrying different dominant markers. These P-1152BX2*, RS3BX2*, and P-1152+RS3BX2* females were crossed with males carrying a BX2 naive cluster and G1 progeny having paternally, but not maternally, inherited a BX2 cluster were crossed to establish lines. These lines carry a second order paramutated locus. (B) These lines were subsequently tested for trans-silencing capacities at generations 1, 4, and 36. The TSE percentage is indicated with the number of egg chambers counted in parentheses. Strong silencing capacities are observed over generations showing that P-1152BX2*, RS3BX2*, and P-1152+RS3BX2* are strongly paramutagenic.

Maternally transmitted small RNAs can affect an adult somatic phenotype

piRNAs are maternally transmitted not only to the posterior pole of Drosophila embryos but also to more anterior regions where they can be implicated in gene regulation. Indeed, piRNAs homologous to transposable elements (roo, 412) have been implicated in nanos mRNA decay at the anterior part of the embryo by a mechanism involving mRNA deadenylation (Rouget et al. 2010). Furthermore, a high number of maternal RNAs were shown to undergo piRNA-dependent destabilization in the soma during maternal-to-zygotic transition in embryos (Barckmann et al. 2015). In addition, piRNAs can have an effect in somatic cells of the gonads. Indeed, in D. simulans, expression of the tirant transposable element in ovarian follicle cells was shown to be affected by tirant homologous piRNA maternal transmission (Akkouche et al. 2013). Finally, maternal depletion of Piwi in D. melanogaster impacted white variegation in adult eyes (Gu and Elgin 2013). Therefore, piRNA maternal deposition can impact not only the germline of progeny but also somatic tissues. During the course of experiments performed to study paramutation in the germline using the T-1 line as paramutagenic line (de Vanssay et al. 2012), we detected impact of P{lacW} homologous small RNA maternal inheritance on somatic tissues, which we further investigated.

The T-1 paramutagenic line, which carries seven P{lacW} copies inserted in tandem (Dorer and Henikoff 1994, 1997), was shown to produce abundant ovarian piRNAs including piRNAs homologous to the white transformation marker (de Vanssay et al. 2012). The white gene is involved in eye pigment transport and its loss of function results in white eyes instead of red ones (Figure 5B). The T-1 cluster shows strong variegation for the white marker, i.e., stochastic ON-OFF white repression from cell to cell (RIGS) (Dorer and Henikoff 1994, 1997). RIGS is associated with heterochromatin formation on tandem repeats, which includes de novo binding of HP1 at the locus (Fanti et al. 2003). In the T-1 line, RIGS-induced variegation was enhanced by X-ray-induced chromosomal rearrangements. We first investigated the potential somatic effects of P{lacW} piRNA maternal inheritance by performing reciprocal crosses between T-1/Cy and white mutant individuals from different genetic backgrounds and comparing female progeny eyes from these crosses (eye images and pigment amounts). Figure 5A shows the very clear difference observed for all the three backgrounds tested (w1118, w1118Cy/Xa, and ywc). Females having maternally inherited the T-1 cluster presented stronger white repression than females having paternally inherited the cluster. Note that in each case the two types of females have strictly the same genotype. The differences were confirmed by analysis of pigment levels (Figure 5A).

Somatic impact of maternally transmitted small RNAs. The effect of P{lacW} small RNA maternal inheritance on P{lacW} cluster expression in adult eyes was tested using two different transgene clusters, T-1 (A) and DX1 (B), as a read-out. T-1, but not DX1, has trans-silencing capacities, correlated with the presence of abundant ovarian piRNAs. The G0 cross is indicated above the picture and was performed at 25°. Green female symbol indicates cytoplasmic inheritance containing P{lacW} piRNAs. The genotype (chromosome 2) of the G1 females analyzed is given below the picture. Cy and Xa are chromosomes devoid of transgenes and carrying dominant markers. The maternally inherited chromosome is written above the bar. (A and B) Pigment levels (absorbance at 480 nm) are shown beside the pictures. Numbers in abscissa indicate the genotype analyzed (reported directly on the head picture). For pigment level dosages, Cantony and w1118 female heads served as WT and w null mutant controls, respectively. Control pictures are boxed in B. Bars indicate the standard error. Comparisons were performed using the Student’s t-test: *P < 0.05; **P < 0.01; ***P < 0001. Maternal inheritance of P{lacW} small RNAs produced by a T-1 female results in increased silencing of both T-1 and DX1 expression in the eye.

We further tested the effect of P{lacW} homologous piRNA maternal inheritance on the eye phenotype in flies carrying another P{lacW} cluster called DX1. This cluster has six tandem P{lacW} copies, one of which is in opposite orientation to the others (Dorer and Henikoff 1994, 1997). This cluster shows a low level of variegation at 25° but variegation is stronger at higher temperatures presenting almost a fully white phenotype at 29° (data not shown). DX1 has no trans-silencing capacities (Ronsseray et al. 2001; de Vanssay et al. 2012). The two reciprocal crosses were first performed between DX1 and w1118; Cy/Xa individuals to test for the existence of a maternal effect of DX1 inheritance on its eye phenotype and no significant difference was observed between the two types of progeny for white expression (Figure 5B). Further DX1 individuals were crossed with T-1 individuals and female progeny having inherited DX1, but not T-1, were analyzed. Figure 5B shows that DX1 females, having maternally inherited T-1 cytoplasm, i.e., containing transgenic small RNAs, presented eyes with a stronger level of white repression than DX1 females having inherited DX1 cytoplasm. Thus, T-1 maternal cytoplasm induced DX1 repression in adult heads. Moreover, females produced by crossing DX1 females and T-1 males showed significantly stronger white repression than those produced by crossing DX1 females and w1118; Cy/Xa males. This likely resulted from genetic background effects since no paternal inheritance from T-1 P{lacW} piRNAs was previously detected (de Vanssay et al. 2012). In contrast, the difference observed between the progeny of reciprocal crosses of DX1 and T-1 individuals can be attributed to piRNA maternal transmission since these two types of female progeny have the same genotype. Consequently, a repressive effect of a T-1 cytoplasm, which carries a high level of P{lacW} piRNAs, can be observed in progeny adult eyes with both the T-1 and DX1 P{lacW} clusters as a read-out. To understand this small RNA somatic impact in progeny, it will be necessary to test it in zygotes, which are mutants for small RNA silencing pathways or chromatin-based repression pathways.

Discussion

The present results establish the paramutated BX2* locus as a typical piRNA dual-strand cluster whose de novo small RNA production involves nuclear and cytoplasmic actors of the piRNA pathway. Among the small RNAs produced by the BX2* locus, the 23- to 28-nt piRNAs, but not the 21-nt RNAs, are required for paramutation. Finally, a stable paramutation can be generated between partially homologous loci that are not allelic.

Implication of the primary piRNA pathway in BX2* capacities

Mutant analysis shows thus that aubergine, rhino, cutoff, and zucchini are required for a paramutated locus to exhibit piRNA production capacities and trans-silencing. Two main molecular models, one nuclear, one cytoplasmic, have been proposed for the paramutation process (de Vanssay et al. 2013; Le Thomas et al. 2014). Considering a cytoplasmic paramutation model, it can be proposed that a trans-generational nuage-to-nuage transfer of information can be the only factor that explains the acquisition of silencing capacities by the BX2* paramutated locus. It was previously shown by qRT-PCR that the BX2 naive (nonparamutated) locus is transcribed in both orientations (de Vanssay et al. 2012). Thus, during paramutation, piRNAs loaded on Aub or Ago3 in the nuage of T-1 or BX2* female nurse cells could be transferred to the nuage of zygotic primordial germ cells (PGCs) having paternally inherited a BX2 naive locus, resulting in de novo targeting of BX2 P{lacW} transcripts by the ping-pong machinery and accounting by itself for the acquisition of silencing capacities by the BX2* paramutated locus. In a nuclear paramutation model, maternally transmitted piRNAs, loaded on Piwi, could enter into PGC nuclei and activate de novo targeting of BX2 naive transcript by the primary piRNA pathway via promoting the binding of the Rhino-Deadlock-Cutoff complex on the P{lacW} cluster. This model is suggested by the fact that deposition of Rhino at several piRNA producing loci depends on Piwi (Mohn et al. 2014). It is also strongly supported by ChIP analysis, which showed that a BX2* cluster, as tested in G1 ovaries of the paramutation experiment, exhibited an increase in H3K9 methylation levels when compared to a naive BX2 locus (Le Thomas et al. 2014). H3K9me3 residues can indeed be targeted by the chromodomain of Rhino, resulting in RDC complex stabilization on the cluster. The mutant analysis reported here provides the functional assay that shows that rhino, cutoff, and zucchini are necessary for BX2* trans-silencing and piRNA production capacities (Figure 1). Therefore, paramutation-linked repressive properties do not solely depend on cytoplasmic ping-pong activation resulting from nuage-to-nuage trans-generational transfer of information. These properties depend also on the primary piRNA pathway, which is linked to the binding of the RDC complex on the locus (Mohn et al. 2014; Zhang et al. 2014). Analysis focused on the T-1 paramutagenic locus, which produces a high level of ovarian P{lacW} piRNAs, strongly reinforces this model (Le Thomas et al. 2014). Indeed, ChIP analysis performed on ovaries of G1 females produced by the two reciprocal crosses between T-1 and wild-type (devoid of transgene) individuals showed that maternal inheritance of the T-1 cytoplasm is associated with an increase of Rhino and Cutoff binding on the T-1 P{lacW} cluster in progeny ovaries (Le Thomas et al. 2014). Paramutation associated repressive properties also depend on the Zuc and on Aub activities (Figure 1), which take place in the nuage. In conclusion, chromatin analyses (Le Thomas et al. 2014) and functional analyses performed with mutants (Figure 1) complement each other to show that trans-generational induction of piRNA-mediated silencing by maternally inherited piRNAs involves nuclear and cytoplasmic steps of the piRNA pathway, including those involved in the atypic transcription of the piRNA loci. Thus, during paramutation, the paramutated locus is modified and remains so over generations.

BX2* transgenic 21-nt RNAs are actual siRNAs whose production also depends on the piRNA machinery

Deep sequencing of ovarian small RNAs associated with both telomeric P-transgene insertions and to the T-1 paramutagenic or BX2* paramutated clusters showed abundant 21-nt transgene homologous RNAs, in addition to 23- to 28-nt piRNAs (de Vanssay et al. 2012). Both sense and antisense 21-nt RNAs were found that contained no ping-pong signatures. These 21-nt RNAs were proposed to be Dicer-2 dependent. Here, ovarian small RNAs of BX2*, Dcr-2−/− females have been analyzed by deep sequencing. As expected, BX2* 21-nt RNAs were almost absent in these Dcr-2 mutant females showing that they are genuine siRNAs (Figure 1). Strikingly, all the piRNA pathway mutations tested also resulted in the loss of the transgene-derived 21-nt RNAs (Figure 1). This suggests that P{lacW} siRNAs could be generated downstream of the piRNA pathway. It was shown further that production of these transgene-derived siRNAs by a female requires piRNA, but not siRNA, maternal inheritance, confirming that their appearance is downstream of piRNAs (Figure 2). The 21-nt small RNA production appears to be a common feature for a number of piRNA-producing loci (Czech et al. 2008; Malone et al. 2009; Pane et al. 2011). cuff mutant analysis was shown to strongly affect 21-nt RNA production by a number of double-strand piRNA-producing loci, including the 42AB locus (Pane et al. 2011; Le Thomas et al. 2014). What mechanism could explain the 21-nt RNA dependence on the piRNA pathway? P{lacW} homologous siRNA biogenesis depends on all the piRNA actors tested, including for example Rhino, which is necessary for production of piRNA precursors; Zucchini, which is responsible for slicing steps in the cytoplasm; and Aubergine, which is an actor of the ping-pong amplification of the pool of piRNA. One possibility is that P{lacW} siRNA production results from the fact that both sense and antisense RNAs, produced by this dual-strand cluster, are concentrated locally due to the piRNA machinery, for example in the nuage, leading to occurrence of double-strand RNAs, which can further be processed by Dcr-2. The piRNA mutants tested could alter the production of the piRNAs associated with Piwi, which are produced downstream of the ping-pong amplification step (Han et al. 2015; Mohn et al. 2015), and consequently reduce a positive feedback in the nucleus of these piRNAs (Mohn et al. 2014). This would reduce deposition of Rhino at several piRNA, producing loci (Mohn et al. 2014) and thus affect dual-strand transcription of the BX2 locus, abolish the sense and antisense RNA concentration step, and siRNA production as well. However, a low level of P{lacW} 21-nt RNA is still detectable in Dcr-2 homozygous mutant females (Figure 1F), which present a ping-pong signature (Figure S3J). If we analyze small RNAs produced by the 42AB locus, which is a typical dual-strand cluster piRNA locus, piRNAs are reduced or disappear in all piRNA pathway mutant backgrounds tested (aub−/−, rhi−/−, cuff−/−, zuc−/−, Figure S1, D, H, J, and L). The 21-nt RNAs with a ping-pong signature also disappear in these mutant backgrounds for the 42AB locus (Figure S3, H, P, T, and X). An intriguing variation is observed in the zuc−/− background (Figure S1L), which showed reduction in piRNA levels and a dramatic increase in 21-nt RNA levels for the 42AB locus. This suggests that in the absence of Zuc, precursor transcripts coming from double-stranded piRNA clusters can be processed into 21-nt RNA instead of piRNAs. This could be achieved by Dcr-2, since this enzyme is responsible for the presence of the majority of the 21-nt RNAs coming from piRNA clusters (see above) and these 21-nt RNAs show no ping-pong signature (Figure S3X). Thus, for a given locus, two 21-nt RNA populations coexist in the Drosophila germline: a high level of Dcr-2-dependent siRNAs showing no ping-pong signature whose production could be in competition with that of piRNAs and a low level of Dcr-2 independent 21-nt piRNAs showing a ping-pong signature.

Spreading of the piRNA production capacity within a piRNA-producing cluster can be completed within three generations

piRNA clusters behave as genomic traps in which new insertions of TEs lead to de novo production of piRNA homologous to the new insertions. The kinetics of appearance of this new piRNA production is not clear, but some functional evidence has been provided from previous studies concerning the repression capacity inheritance of the I-factor and of the P-element (Kidwell et al. 1977; Bucheton 1978; Engels 1979). In both systems, it appears that establishment of repressive capacities, following paternal inheritance of I- or P-elements carrying genomes in a cytoplasm devoid of I- or P-homologous piRNAs, is progressive and require several generations to be completed (Engels 1979, 1989; Bregliano et al. 1980; Finnegan 1989; Rio 2002). For example, experiments were performed with a line [Lk-P(1A)] carrying only two regulatory P-elements inserted in a telomeric strong piRNA-producing locus (TAS) (Coen et al. 1994; Ronsseray et al. 1996). It was shown that paternal transmission of the telomeric P-element locus in a cytoplasm devoid of P-homologous piRNAs, followed by maternal inheritance of the telomeric P-elements in successive generations, requires four generations to progressively establish complete P-repression (Coen et al. 1994). Thus, during these generations, a progressive spreading must occur within the TAS locus for piRNA production capacity, which finally results in strong production of piRNAs homologous, not only to the TAS, but also to the P-element. A spreading process for piRNA production was also observed for the flamenco piRNA-producing locus, which functions in the somatic follicle cells that surround germline cysts (Muerdter et al. 2011). Further, analysis of previously established transgenic lines that carry an I-fragment within a P-transgene have shown that piRNA production capacity can spread within transgenes (Olovnikov et al. 2013). The capacity to produce piRNAs invaded the whole I-derived transgene and reached in some cases flanking sequences. A similar spreading process into flanking sequences was also found for natural transposon insertions (Shpiz et al. 2014). It is possible, however, that spreading processes inside or outside a transgene or a TE involve different mechanisms. However, in these last experiments, the capacity of targeted sequences to maintain piRNA production on their own, in absence of the endogenous homologous piRNA locus that stimulated them, was not tested. In this article, we show that cis-spreading of piRNA production on BX2* is maintained over generations in absence of the original input, the telomeric transgenic locus. In addition, this spreading is completed within a few generations.

What mechanism could be responsible for the spreading process? Very recent studies have shown that piRNAs associated with Piwi can be produced downstream of piRNA generation by ping-pong amplification, which can increase piRNA sequence diversity (Han et al. 2015; Mohn et al. 2015). Indeed, piRNAs associated with Ago3 can target complementary RNA and induce production of phased piRNAs (27 nt) corresponding to the slicing of the 3′ tail of the cleaved target RNAs. These phased “trailer” piRNA, once loaded on Piwi, can potentially return to the nucleus and increase both transcriptional repression of homologous targeted TEs or stimulate binding of Rhino on the piRNA-producing locus. One consequence of such an additional mechanism would be the adjustment of the force of repression to the level of transcription of active copies. Another would be the formation of novel piRNAs, which are cis to the sequence targeted initially by piRNAs. This phenomenon would explain why a P-lacZ-white transcript targeted by P-lacZ homologous piRNAs is able to produce piRNAs homologous to white. However, some observations remain to be explained. First, according to this model, when telomeric P-insertions are paternally introduced in a lineage and thereupon are maternally transmitted, establishment of repression would be expected to require only one generation to be completed but in fact it was shown to be progressive over generations (Coen et al. 1994). It is possible that nuclear reinforcement mediated by trailer piRNAs loaded on Piwi on the piRNA-producing locus is necessary, and this reinforcement involves maternal transmission to be efficient and thus requires more than one generation to be completed. Second, we performed an additional paramutation experiment in which a paternally inherited BX2 locus was introduced in a maternal cytoplasm produced by Lk-P(1A) females. In that experiment, no paramutation of the paternally transmitted BX2 cluster was observed (Figure S6) despite maternal inheritance of P-element homologous piRNAs corresponding to 820 bp of homology of the 10,691 bp of the P{lacW} transgene. It remains possible, however, that production of such trailer piRNAs is limited by the propagation mechanism itself, making it possible to fill a gap of 4 kb (Figure 3) but not a gap of almost 10 kb (Figure S6).

Role of paramutation in TE repression establishment and stabilization in natural populations

The Drosophila genome contains >140 piRNA-producing loci, most of them composed of truncated versions of TEs (Brennecke et al. 2007). How have these loci been activated in natural populations to produce piRNAs? Paramutation may have played a key role in such a process. Once activated in a single fly by an unknown factor, which could be environmental (Schott et al. 2014), a first piRNA-producing locus may have progressively replaced the inactive allele within the population by a paramutation process similar to paramutation of a BX2 naive locus by a BX2* locus (de Vanssay et al. 2012). Further, the first locus activated may have activated other TE-accumulating loci, despite showing only partial homology and being located on a different chromosome, by a paramutation process similar to that of BX2 by P-1152 or RS3 (Figure 3). These secondarily activated sites could maintain TE repression capacities in individuals from the population that did not inherit the first active piRNA locus if this first locus is not fixed in the population. With time, epigenetic communication between TE-accumulating loci may activate all “genetically” competent loci (sufficient number of tandem repeats, bidirectional transcription, etc.). Moreover, these partially homologous paramutations could also have played a role during migration processes between natural populations. Indeed, a fly bearing active loci arriving from one population into another one could activate loci present on the chromosomes of the recipient population, despite the existence of potential partial divergences between TE-accumulating loci in these different populations. Therefore, it is possible to propose that paramutation may play an important role in epigenome shaping of the Drosophila germline, especially because it can occur even when interacting loci partially diverge in their sequence.

Acknowledgements

We thank Doug Dorer, Steve Henikoff, Attilio Pane, Trudi Schüpbach, Paul Macdonald, and the Bloomington Stock Center for providing stocks and FlyBase.org for providing databases. We thank Ritha Zamy for technical assistance, Neel Randsholt and Anne-Marie Pret for critical reading of the manuscript, and an anonymous referee for useful suggestions. This work was supported by fellowships from the Ministère de l’Enseignement Supérieur et de la Recherche to C.H. and A.A.-L. and by grants from the Association de la Recherche contre le Cancer (Fondation ARC, SFI20121205921, SFI20131200470), the Fondation pour la Recherche Médicale (FRM DEP20131128532), the Association Nationale de la Recherche (ANR, project “plastisipi”) to S.R. and C.A., and the University Pierre et Marie Curie (Emergence EME1223) to L.T. The funders had no role in study design, data collection, analysis, decision to publish, or preparation of the manuscript. The authors declare that no competing interest exists.

, 2002Suppression of heterochromatic gene variegation can be used to distinguish and characterize E(var) genes potentially important for chromosome structure in Drosophila melanogaster.Mol. Genet. Genomics266: 922–932.

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